The objective of the proposed research is to obtain quantitative understanding of protein nucleation and post-nucleation growth of small protein crystals (crystallites) in order to: (a) improve crystallization protocols yielding crystals of a structural perfection that matches state-of-the-art X-ray diffraction techniques used in protein structure/function studies and rational drug design; (b) achieve narrow crystallite size distributions, which are essential for steady sustained release of pharmaceutical protein preparations, such as insulin; and (c) find means to regulate protein crystallization in vivo, e.g., the formation of hemoglobin C crystals in red blood cells. The specific aims to be pursued using four model globular proteins are: 1) Obtain insight into the fundamental mechanisms of protein crystal nucleation through studies of the dependencies of the nucleation rate on protein concentration and solution supersaturation. 2) Define and quantify the effects of: (i) soluble additives, biospecific for each protein, in concentrations representative of those commonly occurring in crystallizing protein solutions; and (ii) particulates with known size and concentration. 3) Based on the insight obtained under Aims 1) and 2), demonstrate that protein crystal nucleation can be controlled to achieve improvements in the areas (a)-(c) above. The basic hypothesis underlying this work is that nucleation concepts derived for inorganic systems, subject to modifications for differences in solution interactions and molecular kinetics, can provide guidance in protein nucleation. A key element of the proposed investigation is a novel automated multi-cell microscopy technique for protein nucleation studies. This technique allows direct measurements of the nucleation rates and quantitative correlations to the nucleation conditions. Unlike previous protein nucleation studies, these nucleation rate determinations are not based on any assumptions about the molecular interactions in the crystallization solution.